Location: Crops Pathology and Genetics Research
Project Number: 2032-22000-017-047-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Apr 1, 2020
End Date: Mar 31, 2022
Objective:
Development and evaluation of transgene constructs and plant viral expression vectors for transient production of three therapeutic biologics recombinant parathyroid hormone (PTH) residues 1-34, granulocyte colony stimulating factor (G-CSF), and Trypsin (TRP) in Lactuca sativa (lettuce), for NASA-medically relevant conditions.
Approach:
Synthesis of cDNA constructs for plant viral expression systems, and cloning of transgenes into the viral expression vectors. In addition to PTH, G-CSF and TRP, we will include a fluorescent reporter such as EGFP for visualization of the production dynamics. We will investigate both a positive sense RNA plant virus, Lettuce mosaic virus (LMV), and a DNA virus, Bean yellow dwarf virus (BeYDV) as viral expression systems. We plan to obtain an LMV isolate from USDA-ARS, Salinas, CA, and maintain it in lettuce and Nicotiana benthamiana plants. Primers will be designed to generate overlapping amplicons by RT-PCR. From infected tissue, total RNA is extracted and amplicons are produced by RT-PCR and sequenced following established protocols. Using amplified products and sequence data, we will design additional primers and clone the full-length genome into a low copy number plasmid by In-Fusion HD Cloning Plus and an infectious clone will be assembled. The amino acid sequence of enhanced Green fluorescent protein (EGFP) gene will be engineered to have two cleavage sites recognized by the potyvirus proteases and inserted in frame with the polyprotein right before the NIa-Pro coding region. The infectivity of the LMV cDNA clone will be evaluated by infiltration of the recombinant agrobacteria into leaves of lettuce (a natural host of LMV) and N. benthamiana (an experimental host of LMV used as a control). Systemic infection of the virus and EGFP expression will be monitored. A BeYDV expression vector will be custom synthesized and cloned into a binary vector for expression. In this plasmid vector, the capsid protein gene will be replaced by EGFP gene during the design, and the absence of capsid protein gene will make the virus incapable of horizontal transmission and systemic movement and thus is not expected to pose a risk. The vector DNA will be introduced into lettuce and alfalfa sprouts as described above. Expression of eGFP will be monitored. Once the infectivity of the LMV and BeYDV clones are established, their EGFP expression efficiency will be evaluated by agroinfiltration assays in the presence of an additional silencing suppressor P19 using at least four replicates. Once the need for P19 is ascertained, we will build plasmid constructs to express FDA approved biologics.
